A scanning microscope of the type specified above is used, for example, in fluorescence microscopy, in which fluorescent dyes are stimulated to emit fluorescence radiation with the aid of an illuminating light bundle. In this application, stimulation light in the form of the illuminating light bundle is guided in a scanning movement over the sample. This scanning movement is realized by means of a scanning unit arranged upstream of the objective, said scanning unit generally comprising one or more movable scanning mirrors and a scanning optical system, which directs the illuminating light bundle onto the entry pupil of the objective. The fluorescence radiation stimulated in the sample by the illuminating light bundle is guided back into the scanning microscope by the objective and is fed in the form of a detection light bundle to a detection unit, which generally contains various lenses for shaping and diverting the detection light bundle as well as a detector which finally detects the detection light bundle.
While descanned detectors are conventionally used in confocal microscopy, non-descanned detectors (NDD) are used in other microscopy applications such as, for example, multiphoton microscopy, light sheet or single-plane microscopy or multispot or multi-foci microscopy. A descanned detector firstly receives the detection light bundle after it has been guided back to the scanning unit and has been converted back by said scanning unit into a fixed detection light bundle. A fixed light bundle of this type is also the illuminating light bundle before it strikes the scanning unit. In contrast, a non-descanned detector receives the detection light bundle without this having previously been fed back to the scanning unit. Thus, the detection light bundle arrives at the detector without influencing by the scanning unit.
In multiphoton microscopy, by non-linear effects, only fluorescent dyes in a spatially limited simulating focus, produced by the illuminating light bundle focused on the sample, are stimulated to emit fluorescence radiation. The entire fluorescence radiation which comes from the stimulating focus can now be detected by the non-descanned detector, taking into account the known position of the stimulating focus. Since the illuminating light bundle is guided over the sample in a scanning movement, a three-dimensional sample image can consequently be produced.
Various beam splitters and filters which spectrally influence the detection light bundle in the desired manner are generally located in the detection beam path of a scanning microscope. Thus, beam splitters may, for example, be used to split the detection light bundle into various separate beams depending on the wavelength, which beams are then fed to various detection channels which in each case contain their own detector. The wavelength range to be detected can be determined for each detector by means of optical filters.
In particular, point detectors (for example photomultipliers, avalanche photomultipliers or hybrid photodetectors), line detectors or surface detectors or array detectors (for example CCD, EMCCD, CMOS, sCMOS or QIS (quanta image sensor)) can be used as detectors.
In confocal microscopy, spectral detectors are also increasingly being used which allow the user to freely select the wavelength ranges to be detected before recording the image. In a spectral detector of this type, the detection light bundle is separated, for example by means of a prism, into its spectral proportions and the wavelength ranges to be detected are selected from these.
In contrast, the use of such flexibly usable spectral detectors in non-descanned applications was previously not readily possible as the detection light bundle in the detection unit executes a scanning movement there, which does not occur in the confocal application owing to the return of the detection light bundle to the scanning unit. Non-descanned detectors have therefore hitherto been equipped with conventional interference filters or interference beam splitters, the spectral characteristics of which are fixed, i.e. cannot be varied by the user during the experiment.
It is proposed in DE 10 2006 034 908 A1, in a scanning microscope, to use a spectrally selective component in the form of an edge filter, the limit wavelength of which, also called the spectral edge, varies along the filter (graduated filter). In this case, the spectral edge of the filter separates a wavelength range of the transmission from a wavelength range in which no transmission takes place.
The use of an edge filter of this type with a locally variable spectral edge allows the user to adjust the spectral characteristic of the detector as desired. However, problems also occur here which have been recognized by the present inventors and are not addressed in DE 10 2006 034 908 A1. An adequately steep spectral edge, i.e. an adequately sharp limit wavelength, can only be realized when the detection light bundle striking the filter has an adequately small diameter. Each increase in the bundle diameter inevitably leads to a reduction in edge steepness. The smaller the edge steepness, the less precise is the spectral characteristic of the detector.
Moreover, the spectral edge position of the filter depends on the angle of incidence at which the detection light bundle strikes the filter. The present inventors have recognized that this is disadvantageous, in particular in a non-descanned detector, in which the angle of incidence of the detection light bundle varies as a result of the scanning movement of the illuminating light bundle.